Isolation of species-specific protein antigens of Rickettsia typhi and Rickettsia prowazekii for immunodiagnosis and immunoprophylaxis.
A simple procedure for the selective isolation of the protective species-specific protein antigens (SPAs) of Rickettsia typhi and Rickettsia prowazekii was developed to permit use of the SPAs in the immunodiagnosis and immunoprophylaxis of typhus infections. Although the SPAs were readily extracted from lysozyme- or detergent-treated rickettsiae, as measured by rocket immunoelectrophoresis, other polypeptides were also present, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In contrast, both water and seven buffers, each at a 10 mM concentration and pH 7.6, were nearly equally effective in the selective release of the SPAs from whole cells by extraction for 30 min at 45 degrees C. High-ionic-strength buffers and MgCl2 abolished this SPA release, thus suggesting that divalent cations were important in the binding of the SPAs to the cell envelope. The efficacy of the dilute buffer extraction procedure for isolation of large amounts of SPAs was tested by further characterization of the supernatants obtained by centrifugation (200,000 x g) of two successive tris-(hydroxymethyl)aminomethane-hydrochloride buffer (Tris) extracts. With this procedure, between 10 and 15 mg of SPA was obtained from 100 mg of purified rickettsiae. Although low-molecular-weight ribonucleic acid fragments were released into the Tris extracts in significant amounts, only the SPAs were detected, in significant quantities, as measured by polyacrylamide gel electrophoresis and rocket immunoelectrophoresis. The Tris extracts contained the same major and minor SPA polypeptides as those observed previously in SPA preparations obtained by extensive diethylaminoethyl-cellulose column chromatography, but the Tris SPAs were more satisfactory antigens in an enzyme-linked immunosorbent assay.
D000942:Antigens, Bacterial / Q000032:analysis / Q000302:isolation & purification*; D001426:Bacterial Proteins / Q000276:immunology*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D012283:Rickettsia prowazekii / Q000276:immunology*; D012286:Rickettsia typhi / Q000276:immunology*; D013045:Species Specificity; D014437:Typhus, Endemic Flea-Borne / Q000175:diagnosis* / Q000517:prevention & control; D014438:Typhus, Epidemic Louse-Borne / Q000175:diagnosis* / Q000517:prevention & control
D000942:Antigens, Bacterial; D001426:Bacterial Proteins